HETEROZYGOTE AND MUTATION DETECTION BY DIRECT AUTOMATED FLUORESCENT DNA-SEQUENCING USING A MUTANT TAQ DNA-POLYMERASE

We describe a method for direct cycle sequencing of PCR fragments ampl ified from genomic DNA or cDNA. DNA sequencing template is amplified u sing PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent s equencing primers, Sanger dideoxy sequencing chemistry and an enzyme m ixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase . The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders fr om unpurified PCR fragments of sufficiently high quality such that het erozygotes can be reproducibly detected and identified by software tha t recognizes signal-strength patterns indicative of mixed-base positio ns.